Laccase biomonomer crosslinked reusable cryogenic column for synthesis of novel tyrosine-based crosslinker

This study introduces a reusable cryogenic column system immobilized with laccase enzyme (laccase-M) for the sustainable synthesis of a novel tyrosine-based crosslinker, dityrosine-dimethacrylate (Dityr-diMA). By leveraging methacrylated tyrosine (Tyr-MA) as a biocatalytic platform, the laccase-M column enables efficient polymerization via oxidative coupling, eliminating the need for toxic photoinitiators or metal catalysts. Compared to conventional HEMA-based columns, the laccase-M system achieved a 19.5% higher water retention capacity (819.83% → 980.16% swelling ratio) and maintained 74.02% relative enzyme activity (172.32 → 127.55μMmin^-1) over 50 reuse cycles, with a remarkably low decay rate of 0.52 % per cycle, demonstrating unprecedented operational stability for cryo-immobilized laccase systems. The enzymatic method also reduced organic solvent consumption by 60 %. Structural analyses (SEM, BET) revealed that the crosslinked Dityr-diMA network exhibits a homogeneous pore architecture (BET surface area: 6.219m²g^-1) and enhanced material stability. Kinetic studies confirmed the system's superior substrate affinity, with a 10-fold decrease in Km values (0.0192 → 0.0021 mM), highlighting its industrial scalability. Compared to UV crosslinking methods (Eosin Y, Irgacure 2959, LAP) and (metal complexes ([Ru(II)(bpy)₃]²⁺)), the enzymatic approach provided a controlled polymerization mechanism with low cytotoxicity. This study demonstrates with experimental data that enzymatic crosslinking can be used as a non-toxic, scalable, and efficient strategy in the design of high-performance biomaterials; It offers an environmentally friendly alternative to traditional methods, paving the way for safer and more sustainable approaches in biomedical and tissue engineering applications.